*3.3. Replacement of SENP*

Again, we used the LeishGedit toolbox to design 5′ - and 3′ -sgRNAs. Selection marker cassettes were amplified from the pTPURO and pTBLAST plasmids with ends targeting the SENP UTR sequences (Figure 3A). A mix of amplified sgRNA coding DNA and amplified selection marker cassettes was then transfected into *L. donovani* (Cas9/T7-RNAP). The transfectants were then cultivated under puromycin/blasticidin double selection. Selected parasite populations were then subjected to limiting dilution to raise putative SENP−/<sup>−</sup> clones. RT-qPCR analysis of SENP RNA confirmed the lack of GOI-specific RNA for all selected clones, confirming them as null mutants (Figure 3B). Reintroduction of SENP as an episomal gene copy into clone#1 resulted in a massive over production of SENP RNA (SENP−/−/+, Figure 3B). Given the confounding potential of Cas9 expression in the mutants, we analyzed them for Cas9 RNA as well (Figure 3C). Only *L. donovani* (Cas9/T7-RNAP) kept under the episome-specific antibiotic selection showed detectable levels of Cas9 RNA while the SENP−/<sup>−</sup> mutants had lost the expression plasmid during selection and cloning.

To confirm the loss of SENP on a genomic level, we also performed whole genome sequencing of genomic DNA (gDNA) from *L. donovani* wild type, *L. donovani* (Cas9/T7-RNAP), *L. donovani* SENP−/<sup>−</sup> cl.1 and *L. donovani* SENP−/<sup>−</sup> cl.2. Next generation sequencing reads were then aligned to *L. donovani* chromosome 26, using the Bowtie2 algorithm. As expected, both wild type and the Cas9/T7 strain showed uninterrupted read coverage over the SENP gene locus. Conversely, the SENP−/<sup>−</sup> cl.1 showed a complete lack of SENP-specific reads, while clone 2 showed minimal read coverage, possibly indicating a mosaic population (Figure 3D). However, RT-qPCR analysis (Figure 3B) did not show a low level SENP RNA presence. Still, we chose clone 1 for our further analyses.
